Hybridization Probes
The RNA or DNA for many microorganisms and viruses have been isolated and sequenced. Nucleic acid probes are currently available to identify a large number of infections. Nucleic acid probes are detectable nucleic acid sequences that hybridize to complementary RNA or DNA sequences in a test sample. Detection of the probe indicates the presence of a particular nucleic acid sequence in the test sample for which the probe is specific. In addition to aiding scientific research, DNA or RNA probes can be used to detect the presence of viruses and microorganisms such as bacteria, yeast and protozoa as well as genetic mutations linked to specific disorders in patient samples. Grunstein et al., Proc. Natl. Acad. Sci. USA 72:3961 (1975) and Southern, J. Mol. Biol. 98:503 (1975) describe hybridization techniques using radiolabelled nucleic acid probes. Nucleic acid hybridization probes have the advantages of high sensitivity and specificity over other detection methods and do not require a viable organism. Hybridization probes are often labelled with a radioactive substance that can be easily detected. A radioactive hybridization assay for HPV is currently available in the form of the ViraType.TM. or ViraPap.TM. kit by Digene Diagnostics (Silver Spring, Md.).
The existing hybridization techniques that utilize radioisotopes to label probes introduce additional expenses for disposal of radioactive waste products and monitoring of personnel and the workplace for contamination. In addition, the short half-life of radioactive compounds such as .sup.32 P requires that radioactive probes be produced frequently. Radioactive nucleic acid hybridization is therefore discouraged in commercial areas such as clinical diagnosis.
Probes have been indirectly labelled in an attempt to avoid the problems associated with direct radioactive labelling. The most common method of indirect labelling is to attach biotin, a small vitamin, to the nucleic acid using a chemical or enzymatic technique. Following hybridization, the biotin is detected by reaction with avidin, an egg white protein which has been labelled with an enzyme or fluorochrome. Bound enzyme can be detected by reaction with color-producing substrates and the fluorochrome can be seen when reacted with incident light of appropriate wavelength. Indirect labelling of hybridization probes with biotin or other haptens often increases the "hydrophobicity" of the probe. The probe tends to interact non-specifically with materials other than the complementary nucleic acid target, leading to high background. High background reduces sensitivity and increases the likelihood of a false-positive result. Indirect labelling is also less sensitive than direct labelling because the labelling density is limited; only a small fraction of the bases are labelled giving a limiting number of sites for signal generation. An increase in the labelling density of a probe leads to increased non-specific binding, higher background, and ultimately, failure of the probe to hybridize with its target due to the interference of the hapten with base pairing. Indirectly labelled probes are therefore not well suited to clinical diagnosis.
Hybridization has been detected with the use of an intercalating agent such as acridine orange or ethidium bromide as described in U.S. Pat. No. 4,563,417 to Albarella et al. The intercalating agent becomes inserted between hybridized base pairs of probe and sample nucleic acids and causes the tertiary structure of the helix to unwind. An antibody specific for the newly formed antigenic determinant created by the intercalating agent and the unwound helix is detected by conventional means. This method lacks selectivity for the target hybrids because intercalating agents fail to recognize specific sequences. Furthermore, the antibodies recognize only the intercalating agent/nucleic acid complex, but do not detect a specific sequence. Therefore, additional selection or purification steps are required to prevent non-specific signal, making this approach poorly suited for clinical diagnosis.
Hybridization can also be detected with the aid of an antibody specific for a labelled probe as described in U.S. Pat. No. 4,743,535 to Carrico. The probe is labelled with a detectable substance such as flavin adenine dinucleotide (FAD) or a fluorescent agent. An antibody specific for the labelled probe, after it has hybridized to the sample nucleic acid, is detected by a biochemical reaction. This method of detection also creates non-specific binding and the likelihood of false-positive results and is not well suited for clinical screening.
Monoclonal antibodies to DNA-RNA hybrids are now available. U.S. Pat. No. 4,732,847 to Stuart et al. and the publication of Stuart et al., Proc. Natl. Acad. Sci., USA 78:3751 (1981) describe a method of hybridization detection involving a monoclonal antibody specific for a poly(A)-poly(dT) duplex. A monoclonal antibody specific for DNA-RNA hybrids, secreted by hybridoma HB 8730, is disclosed in U.S. Pat. No. 4,833,084 to Carrico et al. The isolation of anti-DNA-RNA hybridomas has improved the development of assays for genetic mutations linked to specific defects and the detection of bacterial and viral infections. However, assays utilizing these anti-hybrid monoclonal antibodies often suffer from a high level of non-specific binding causing false positive results. Boguslawski et al., J. Immunol. Methods 89:123-130 (1986) developed a hybridization assay using anti-hybrid coated polystyrene beads isolated on filter paper in an attempt to reduce non-specific binding and avoid complicated washing procedures.